Abstract
To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets first observable after 1 h at 32°C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16–p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process.
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Acknowledgements
We thank Drs G Poirier, I Schmitz, B Voitesek, and X Wang for supplying antibodies to PARP, caspase 8, p53 and APAF. We are greatful to Drs J Pietenpol and N Tsuchida for providing BCL-2-, VP16-p53-, and p53val138 expression plasmids. This study was supported in part by grant P12814-GEN from the Austrian Science foundation to H Kovar and by private donations to the Children's Cancer Research Institute.
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Kovar, H., Jug, G., Printz, D. et al. Characterization of distinct consecutive phases in non-genotoxic p53-induced apoptosis of Ewing tumor cells and the rate-limiting role of caspase 8. Oncogene 19, 4096–4107 (2000). https://doi.org/10.1038/sj.onc.1203780
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DOI: https://doi.org/10.1038/sj.onc.1203780
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